Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

The Synthesis of the Sixteen Possible 2′-O-Methyl MMI Dimer Phosphoramidites: Building Blocks for the Synthesis of Novel Antisense Oligonucleotides

Abstract

The synthesis of Methylene(methylimino) or MMI linked nucleoside dimers in all sixteen possible configurations has been accomplished via a reductive coupling of a nucleosidic aldehyde with an hydroxylamine. This has allowed us to prepare all of the necessary 2′-O-methyl MMI dimer building blocks necessary for use in an antisense motif.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Role of evolutionary and ecological factors in the reproductive success and the spatial genetic structure of the temperate gorgonian Paramuricea clavata

Dispersal and mating features strongly influence the evolutionary dynamics and the spatial genetic structure (SGS) of marine populations. For the first time in a marine invertebrate, we examined individual reproductive success, by conducting larval paternity assignments after a natural spawning event, combined with a small‐scale SGS analysis within a population of the gorgonian Paramuricea clavata. Thirty four percent of the larvae were sired by male colonies surrounding the brooding female colonies, revealing that the bulk of the mating was accomplished by males from outside the studied area. Male success increased with male height and decreased with increasing male to female distance. The parentage analyses, with a strong level of self‐recruitment (25%), unveiled the occurrence of a complex family structure at a small spatial scale, consistent with the limited larval dispersal of this species. However, no evidence of small scale SGS was revealed despite this family structure. Furthermore, temporal genetic structure was not observed, which appears to be related to the rather large effective population size. The low level of inbreeding found suggests a pattern of random mating in this species, which disagrees with expectations that limited larval dispersal should lead to biparental inbreeding. Surface brooding and investment in sexual reproduction in P. clavata contribute to multiple paternity (on average 6.4 fathers were assigned per brood), which enhance genetic diversity of the brood. Several factors may have contributed to the lack of biparental inbreeding in our study such as (i) the lack of sperm limitation at a small scale, (ii) multiple paternity, and (iii) the large effective population size. Thus, our results indicate that limited larval dispersal and complex family structure do not necessarily lead to biparental inbreeding and SGS. In the framework of conservation purposes, our results suggested that colony size, proximity among colonies and the population size should be taken into consideration for restoration projects.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

1,25-Dihydroxyvitamin D3 receptor in bovine thymus gland

1,25-Dihydroxyvitamin D₃ [1,25-(OH)₂D₃] receptor was characterized after partial purification of thymus cytosol by ammonium sulfate fractionation. The 1,25-(OH)₂D₃ receptor sediments at 3.7S in 5–20% sucrose gradients. The binding of 1,25-(OH)₂D₃ in thymic cytosol was a saturable process with high affinity (Kd = 0.12?0.48 nM) at 4°C. Competition for 1,25-(OH)₂[³H]D₃ receptor by nonradioactive analogs demonstrated the affinities of these analogs to be in order; 1,25-(OH)₂D₃ = 1,24R,25-(OH)₃D₃ = 1,25S,26-(OH)₃D₃ = 1,25R,26-(OH)₃D₃ > 1,25-(OH)₂D₃-26,23 lactone > 25-OHD₃ > 23R,25-(OH)₂D₃ > 24R,25-(OH)₂D₃ > 23S,25-(OH)₂D₃ ? 25-OHD₃-26,23 lactone. The receptor bound to DNA cellulose columns in low salt buffer and eluted as a single peak at 0.21 M KCl. These findings provide evidence that the thymus possesses a 1,25-(OH)₂D₃ receptor with properties indistinguishable from 1,25-(OH)₂D₃ receptors in other tissues.     

Sequence analysis of the grey mouse lemur (<Emphasis Type="Italic">Microcebus murinus</Emphasis>) <Emphasis Type="Italic">MHC class II DQ</Emphasis> and <Emphasis Type="Italic">DR</Emphasis> region

We here report the genomic organisation of the grey mouse lemur (Microcebus murinus) MHC class II DQ and DR region based on BAC clone analysis. The sequenced Mimu-MHC haplotype spans 343 kb and encompasses the genes TAP2, DOB, DQB, DQA, DRB, DRA, BTNL2 and a further BTNL gene. The DQ and DR genes of this haplotype are not duplicated. Mimu-DOB is not transcribed and represents a pseudogene due to deletions and premature stop codons. Analysis of BAC clone DNA, a cDNA sample and eight genomic DNA samples suggests that Mimu-DRB, Mimu-DQA and Mimu-DQB are highly polymorphic with the majority of peptide-binding residues being affected by polymorphisms. In contrast, Mimu-DRA is moderately polymorphic, and the variable amino acid positions are not part of the peptide-binding region. Phylogenetic analysis of Mimu-DQA and Mimu-DQB and other primate DQA and DQB genes indicates that duplication of DQA and DQB loci occurred in Anthropoidea after the split from Strepsirrhini.     

Characterization of the role of the receptors PEX5 and PEX7 in the import of proteins into glycosomes of Trypanosoma brucei

Peroxins 5 and 7 are receptors for protein import into the peroxisomal matrix. We studied the involvement of these peroxins in the biogenesis of glycosomes in the protozoan parasite Trypanosoma brucei. Glycosomes are peroxisome-like organelles in which a major part of the glycolytic pathway is sequestered. We here report the characterization of the T. brucei homologue of PEX7 and provide several data strongly suggesting that it can bind to PEX5. Depletion of PEX5 or PEX7 by RNA interference had a severe effect on the growth of both the bloodstream-form of the parasite, that relies entirely on glycolysis for its ATP supply, and the procyclic form representative of the parasite living in the tsetse-fly midgut and in which also other metabolic pathways play a prominent role. The role of the two receptors in import of glycosomal matrix proteins with different types of peroxisome/glycosome-targeting signals (PTS) was analyzed by immunofluorescence and subcellular fractionation studies. Knocking down the expression of either receptor gene resulted, in procyclic cells, in the mislocalization of proteins with both a type 1 or 2 targeting motif (PTS1, PTS2) located at the C- and N-termini, respectively, and proteins with a sequence-internal signal (I-PTS) to the cytosol. Electron microscopy confirmed the apparent integrity of glycosomes in these procyclic cells. In bloodstream-form trypanosomes, PEX7 depletion seemed to affect only the subcellular distribution of PTS2-proteins. Western blot analysis suggested that, in both life-cycle stages of the trypanosome, the levels of both receptors are controlled in a coordinated fashion, by a mechanism that remains to be determined. The observation that both PEX5 and PEX7 are essential for the viability of the parasite indicates that the respective branches of the glycosome-import pathway in which each receptor acts might be interesting drug targets.     

Honeybee Colony Vibrational Measurements to Highlight the Brood Cycle

Insect pollination is of great importance to crop production worldwide and honey bees are amongst its chief facilitators. Because of the decline of managed colonies, the use of sensor technology is growing in popularity and it is of interest to develop new methods which can more accurately and less invasively assess honey bee colony status. Our approach is to use accelerometers to measure vibrations in order to provide information on colony activity and development. The accelerometers provide amplitude and frequency information which is recorded every three minutes and analysed for night time only. Vibrational data were validated by comparison to visual inspection data, particularly the brood development. We show a strong correlation between vibrational amplitude data and the brood cycle in the vicinity of the sensor. We have further explored the minimum data that is required, when frequency information is also included, to accurately predict the current point in the brood cycle. Such a technique should enable beekeepers to reduce the frequency with which visual inspections are required, reducing the stress this places on the colony and saving the beekeeper time.